Cytokeratin-8 (CK8) is a protein component of the intermediate filaments of the epithelial cell cytoskeleton.
Various studies have shown that CK8 protein is present associated to the plasma membrane of various cancer cells. For example, Godfroid et al. (Journal of Cell Science, 99: 595-607, 1991) described the presence of CK8 proteins on the surface of cultured breast cancer cells. Hembrough et al. (Journal of Cell Science, 108: 1071-1082, 1995) described the presence of CK8 or a CK8-like protein on the surface of hepatocytes, HepG2 cells and breast cancer cells.
CK8 protein has also been detected on the surface of carcinomas of other cancers, such as upper digestive tract cancer (Gires et al., Biochemical and Biophysical Research Communications, 328: 1154-1162, 2005). It has also been shown that CK8 protein is exposed on the surface of invasive and non-invasive tumor cells in colorectal cancers (WO2010/136536). More particularly, it has been shown that in colorectal cancers the appearance of invasive and/or metastatic cells is accompanied by cleavage of human CK8 protein exposed on the surface of these cells. The N-terminus portion of CK8 protein remains exposed on the surface of colon adenocarcinoma cells whereas the C-terminus portion of the protein partially or completely disappears from the surface of these cells.
Colorectal cancer is a particularly invasive cancer. Although significant improvements have been made in the treatment of metastatic colorectal cancer (CRC) patients, this type of cancer remains a significant global public health problem (Weitz et al. Lancet 2005; 365: 153-65). CRC is the third most diagnosed cancer in both sexes with an estimated 940,000 cases per year and the second most common cause of cancer deaths with about 500,000 cases per year (Weitz et al. Lancet 2005; 365: 153-65). In France, 37,000 new patients are diagnosed with CRC each year, of which about half will die of the metastatic disease. However, about 50% of the 36,000 new patients already have metastases at the time of diagnosis or will develop metastases (Weitz et al. Lancet 2005; 365: 153-65; Cunningham et al. Lancet 2010; Chevreul Eur J Health Econ 2010).
The development of more effective drugs (Irinotecan, Oxaliplatin) has made it possible to significantly improve treatments for CRC patients. The last decade has also seen the emergence of molecule-based therapies—monoclonal antibodies (Mab)—directed against specific targets overexpressed (EGF or VEGF receptors) in tumor cells relative to healthy cells. These molecules significantly improve treatments.
Cytokeratin 8 exhibits multiple functions, such as for example the modulation of cell invasion and/or cell apoptosis on the basis of its unique structural hallmark. Related to the cell invasion, CK8 binds plasminogen and tissue-type plasminogen activator (t-PA) and accelerates plasminogen activation on cancer cell surfaces (J Protein Chem. 1998 November; 17(8):845-54). The plasminogen-binding site is located at the C-terminus of CK8.
In addition, by interfering with the plasminogen system, CK8 could be involved in the modulation of the outside-in signal transduction of cancer cells in response to micro-environmental changes (Deryugina and Quigley, J. Biomed Biotechnol, 2012; 2012:564259).
Related to cell apoptosis, the cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and procaspase-9, (Apoptosis (2006) 11:1561-1572). It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced auto cleavage and activation of procaspase-9 in close association with caspase-3.
Various monoclonal antibodies binding to human CK8 protein have been described. In particular, Erlandsson et al. (J. of Molecular Recognition, 16: 157-163, 2003; Johansson et al. Cancer Res 1999, 59, 48-51) described the monoclonal antibody TS1 directed against human CK8 protein, its anti-idiotype αTS1, and the use thereof in immunotherapy for treating carcinomas. Doljak et al. (Cancer Letters, 267: 75-84, 2008) described a monoclonal antibody binding to the VKIALEVEIATY motif conserved among various cytokeratins. This monoclonal antibody binds in particular to CK2, CK8, CK10 and CK18 proteins in MCF-7 breast cancer cells. This antibody inhibits activation of plasminogen on MCF-7 cells in vitro. The antibody M20 marketed by Sigma® was described by Van Muijen, G. et al. (Lab. Invest., 57: 359, 1987; Waseem et al. Biochemistry 2004, 43, 1283-1295). It has been shown that this antibody, specifically binding to CK8 protein, inhibits the growth and the invasive capacity of colorectal cancer tumor cells in tests conducted in vitro and in vivo (WO2010/136536). In particular, in the international patent application WO2010/136536, it was shown that this antibody specifically binds to the uncleaved portion of human CK8 protein present on the surface of colon adenocarcinoma tumor cells. The purified antibody M20 was produced by mouse ascitic fluid. No humanized Mab format has been described for this particular antibody.
Although progress has been made in the identification of antigens on the surface of tumor cells and in the understanding of the mechanisms associated with the development of cancers, and although this progress has enabled the development of monoclonal antibodies that can significantly improve treatments, the effectiveness and the specificity of said monoclonal antibodies can be further improved. Indeed many of the proteins used for the development of targeted therapies are over-expressed in cancer cells but are also expressed, albeit at different levels in healthy tissues. Therefore drugs targeting for a protein key epitope that is more specifically expressed at the cell surface in cancer tissue represent a very promising approach to improve the cancer specificity of the anti-cancer treatments. Thus, there remains a need to develop therapeutic means for treating tumors whose cells express externalized CK8 (eCK8) protein on their surface, in particular for treating colorectal cancers for example. More particularly, there remains a need to develop monoclonal antibodies not only targeting CK8 but more specifically targeting a cancer related epitope that interferes with the tumorigenesis capacities of the cells by modulating well described essential processes of tumor progression such as those involved in cell invasion and cell apoptosis.